Epidemiology and antibiotic resistance of staphylococci on commercial pig farms in Cape Town, South Africa

Staphylococci are responsible for a wide range of infections in animals. The most common species infecting animals include Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus intermedius. Recent increases in antibiotic use and antibiotic resistance in animals highlight the need to understand the potential role of commercial livestock as a reservoir of staphylococci and antibiotic resistance genes. Nasal swabs were collected from 143 apparently healthy pigs and 21 pig farm workers, and 45 environmental swabs of feed and water troughs, from two commercial pig farms in the Western Cape, South Africa. Staphylococci were isolated, identified using mass-spectrometry, and antimicrobial susceptibility testing and Illumina whole genome sequencing were performed. One hundred and eighty-five (185) Staphylococcus spp. isolates were obtained, with Mammalicoccus sciuri (n = 57; 31%) being the most common, followed by S. hyicus (n = 40; 22%) and S. aureus (n = 29; 16%). S. epidermidis was predominantly identified in the farm workers (n = 18; 86%). Tetracycline resistance was observed across all species, with rates ranging from 67 to 100%. Majority of M. sciuri isolates (n = 40; 70%) were methicillin resistant, with 78% (n = 31) harbouring mecA. M. sciuri isolates had genes/elements which were associated with SCCmec_type_III (3A) and SCCmec_type_VIII(4A) and were mostly observed in ST61 strains. ST239 strains were associated with SCCmec_type_III(3A). High rates of tetracycline resistance were identified among staphylococci in the pig farms in Western Cape, South Africa. This highlights the need for policy makers to regulate the use of this antibiotic in pig farming.


Study Population and sample collection
The study population consisted of the pigs on the farms as well as farm workers.Samples were collected from all farm workers who have direct or indirect contact with the pigs and gave consent.Bio-Cult Amies transport swabs with charcoal (Liofilchem, Italy) were used to obtain nasal swabs from farm workers (self-collected from the anterior nares), as well as for sampling the nostrils of the pigs.At least 10 pigs were sampled per pen/holding on each farm.Environmental samples were collected from feed, water troughs and sewage at each farm.Demographic data such as age, sex, antibiotic use and living conditions were collected from the farm workers.The farm managers provided information on antibiotic use and hygiene practices on the farms (Supplementary S1).

Isolation and identification of staphylococci
The swabs were stored at 4 °C for ≤ 4 days before analysis.The swabs were inoculated in 2 ml Lysogeny Broth and incubated aerobically for 6 h at 37 °C to promote the growth of staphylococci.The broth was then sub-cultured onto 5% sheep blood agar plates supplemented with 10 μg/ml each of nalidixic acid and colistin (Thermofisher, USA) to suppress the growth of gram-negative bacteria.Isolates were selected based on a mostly creamy colony morphology consistent with staphylococci.Multiple isolates were collected from each individual, an approach that enabled us to describe the distribution of different staphylococcal species colonizing the pigs and the farm workers.
Presumptive staphylococci were further confirmed based on a negative bile esculin agar test and a positive catalase test (both from NHLS Media Laboratory, Greenpoint, South Africa), and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; VITEK® MS, BioMérieux, France).
Susceptibility testing results were interpreted using the Clinical and Laboratory Standards Institute (CLSI) guidelines, 2020 for staphylococci; but where available, species specific breakpoints were used, such as for S. aureus and S. epidermidis 20 .The term non-susceptible used in this study refers to the CLSI guidelines for resistant and intermediately resistant staphylococci.Multidrug resistance (MDR) is defined as nonsusceptibility to at least one agent from at least three different antibiotic classes 21 .

Statistical analysis
The minimum number of samples to be collected (sample size) was estimated to be 163 using a 95% confidence level, an expected methicillin resistance prevalence of 12% 19 and desired precision of 5% 30 in order to describe the prevalence of methicillin-resistant Staphylococcus spp.Differences in the prevalence of SOSA species and antibiotic resistance rates between the two farms were calculated using Fisher exact and Chi squared tests where appropriate.Microsoft Excel was used for the analysis and p-values of < 0.05 were considered significant.

Ethical considerations
Ethical approval was obtained from the Research Ethics Committee at Stellenbosch University.Informed consent was obtained from the farm workers and managers.The study was approved by the South African Department of Agriculture, Land Reform and Rural Development.
The study was conducted in accordance with the Declaration of Helsinki (2013), as well as South African and ICH GCP Guidelines and the Ethical Guidelines of the Department of Health.The study also observed relevant regulations by the South African Department of Agricultural, Land Reform and Rural development.

Results
On farm A, nasal swabs were collected from nine (9) farm workers and 73 pigs, and 22 environmental samples were taken; while on farm B, nasal swabs were taken from 12 farm workers and 69 pigs, and 23 environmental swabs were collected.
In total, 142 pigs were sampled, with ages ranging from 7 to 35 weeks.All pigs on farm A were finishers aged 25-30 weeks while in farm B, 45 (34.5%) pigs were finishers, 10 weaners aged 7-14 weeks, and nine piglets aged 3-5 weeks (Supplementary S2).Both farms have concrete flooring and engage in the following practices: use commercial food, use antibiotics to prevent infections, health check animals at least every three months, diagnose the animals using farm workers and a veterinarian, and impregnate pigs using artificial insemination.Twenty-one (21) farm workers were included of whom 71% (n = 15) were male.The time workers spent on the farms ranged from 6 to 12 h per day (Supplementary S3).
The prevalence of any staphylococcus was higher on farm A compared to farm B (p = 0.008), however there were no statistically significant differences in the distribution of specific staphylococcal species between farms.This might be due to the small sample size.S. epidermidis was most common amongst farm workers (86%; n = 18) while M. sciuri (33%; n = 47) and S. hyicus (26%; n = 37) were common in pigs.S. aureus, and M. sciuri were common species isolated in the pigs, the farm workers and the environment.In isolates from the pigs, the rate of non-susceptibility to erythromycin ranged from 51 to 100%, to clindamycin from 59 to 100%, and to tetracycline from 93 to 100% between species (Table 2).
A high proportion of the farm workers (n = 12; 57%) were also colonized by tetracycline non-susceptible S. epidermidis.Although only a small number of staphylococcal isolates were isolated from the environment, all isolates of all species were non-susceptible to tetracycline.Supplementary S4 shows the proportion of antibiotic resistance in SOSA isolated from farm workers and the environment.

Whole genome sequence analysis of Staphylococcus spp.
The WGS of four S. aureus and one S. hyicus isolates had insufficient data and were excluded from further analysis, leaving 181 analysable isolates.Speciation of all isolates was confirmed by WGS analysis, except 3 of the 15 S. haemolyticus isolates which were identified as Staphylococcus borealis by WGS.The average nucleotide identity (ANI) of the three isolates was ≥ 95% when compared to S. borealis and ≤ 91% when compared to S. haemolyticus.

Sequence typing and staphylococcal cassette chromosome mec typing
ST1 was the most common sequence type amongst S. aureus (n = 10; 61%) in farm A, while ST9 and ST398 (n = 3; 27% each) and one ST508 were identified in farm B (Supplementary S6).Six additional S. aureus sequence types were identified on the farms, including a novel ST, ST8620 (n = 3; 27%) on farm B (Supplementary S6).
ST61 (n = 11; 31%) was the most common sequence type amongst M. sciuri in farm A and ST239 (n = 15; 68%) in farm B. Other sequence types identified in M. sciuri included eight novel sequence types.All ST61 and ST239 strains harboured mecA.Among the other SOSA species, there was a diversity of STs, with a number of novel STs identified in all species; specifically, all the STs amongst the S. chromogenes isolates were novel.In total, 29 novel sequence types were identified in this study (Supplementary S6).There is no MLST scheme for S. hyicus.

Discussion
This study describes the species distribution, antibiotic resistance profiles and strain types in Staphylococcus spp.from two commercial pig farms in the Western Cape, South Africa.The staphylococcal isolates from this study are carriage isolates from asymptomatic animals, farm workers and environmental isolates.www.nature.com/scientificreports/There was high diversity of staphylococcal species identified, with M. sciuri and S. hyicus as the most frequently isolated species from pigs.Certain species of Staphylococcus, most commonly S. hyicus, are known to cause exudative epidermitis in pigs 31 .Although only a small number of staphylococci were isolated from farm workers and the environment, 18 of the 21 S. epidermidis were from humans.This is similar to a reports in literature that S. epidermidis is the most common SOSA colonizing healthy individuals 32 .This might be an indication that farm workers were colonized outside of the farm setting and that there is a limited transmission of S. epidermidis from workers to the pigs.
The rate of colonisation with S. aureus in the pigs (11%) is similar to what has been reported elsewhere in South Africa (12%) but higher than reports from Nigeria (5%) and Tanzania (4%) 19,33,34 .The rate of S. aureus identified in the farm workers (28%), the environment (17%) and the pigs (11%) confirms what has been described in literature that up to 30% of the human population are asymptomatic carriers of S. aureus and are permanently colonised 35 .The proportion of S. aureus isolates identified in this study also agrees with a systematic review conducted in Africa which reported that the rate of S. aureus in pigs ranges from 0 to 55% 36 .However, data on the environment is lacking.
The most common SOSA identified in pigs were M. scuiri (33%) and S. hyicus (26%).The prevalence of M. sciuri was comparable to a study from Denmark conducted using nasal samples from pigs (40%) 37 .A recent study in Italy also identified M. sciuri as a predominant SOSA species in five pig farms 38 .In contrast, prevalence of S. hyicus in this study was higher than in pigs in studies from Denmark (16%) and Uganda (6%) 37,39 .This is concerning since S. hyicus is known to cause exudative epidermidis in pigs 40 .However, further research is required to understand the high rate of S. hyicus colonisation in the pigs in our setting.Although, no S. hyicus was identified in humans, the three isolated from the environment could be an indication of dissemination from pigs into the environment.
The prevalence of S. aureus including MRSA in pigs in Africa ranges from 0 to 55% 41 .In this study, only one S. aureus (7%) isolate was methicillin resistant.The rate observed in this study is surprisingly lower than a previous report from South Africa by Van Lochem et al., 19 .The high rate of resistance to erythromycin (80%), clindamycin (100%) and tetracycline (93%) in S. aureus in this study and the high erythromycin (89%), clindamycin (89%) and tetracycline (89%) resistance rates seen in isolates from the environment might be indicative of spread from pigs to the environment.The rate of erythromycin, clindamycin and tetracycline resistance among pigs in this study is higher than that described in a review conducted by Katakweba et al., describing antibiotic-resistant S. aureus in pigs in Africa and a nationwide pig study in Switzerland 42 .Interestingly, only one S. aureus isolate from the farm workers was non-susceptible to the above antibiotics, which suggests that transfer of resistant organisms from animals to humans is uncommon.It is also not clear what factors may influence the risk of zoonotic transfer.
While S. aureus is the most studied staphylococcal species, there is limited data on antibiotic resistance in SOSA globally and in Africa.The rate of methicillin resistance in M. sciuri isolates in this study (72%) is lower than what has been reported in Italy (88%) 38 and in broilers in Turkey (100%) 43 .Clindamycin, erythromycin and tetracycline resistance rates of greater than 90% were observed in M. sciuri, consistent with the 99% clindamycin resistance rate reported in M. sciuri from Belgium but higher than the rates of erythromycin (52%) and tetracycline (62%) resistance reported by the same Belgian study 44 .The tetracycline resistance rate was also higher than the 29% reported in Brazilian pigs in 2012 45 .All M. sciuri isolates from the environment were resistant to clindamycin, erythromycin, and tetracycline.This suggests that M. sciuri might be a reservoir of antibiotic resistance genes, and may also indicate movement of isolates from pig to environment (or vice versa).
The high tetracycline resistance rates seen in this study may reflect the use of this antibiotic in pigs in South Africa 46 .Although we could not obtain data on antibiotic usage from the farms in this study, national surveillance data shows that tetracycline is used in large amounts in animals in South Africa; 45% of antimicrobial sales in livestock industry in 2020 were tetracyclines 47 .A recent study by Ocloo et al. showed that 39% of S. haemolyticus carriage isolates were resistant to tetracycline, suggesting potential spillage from pigs or livestock 48 .
A study including three countries in Europe showed that 59% of S. hyicus from Danish pigs were erythromycin resistant which was the highest in Europe 49 , and is similar to the 51% reported in this study.The rate of clindamycin resistance was 59% among S. hyicus isolates, a similar proportion (53%) was reported in diseased pigs in Europe 49 .Clindamycin is used in both clinical and veterinary medicine, making the 100% resistance rate among M. sciuri isolates particularly concerning.The primary cause of clindamycin resistance in M. sciuri is the presence of the sal(A) gene.This gene has been demonstrated to provide intrinsic resistance to both lincosamides and streptogramin A in M. sciuri 50 .In South Africa, fusidic acid is an antistaphylococcal agent used in the treatment of infections due to staphylococci, it is worrying that 91% of M. sciuri isolates were resistant to the antibiotic.
There was only a 60% correlation between phenotypic methicillin resistance and the presence of the mecA gene in SOSA.Additionally, the mecA gene was observed in only one methicillin susceptible S. aureus.No other mec genes were detected in the one MRSA isolate.Similarly, none of the methicillin resistant S. aureus isolated from pigs in Tanzania harboured mecA 33 .In S. epidermidis methicillin resistance was only seen in one isolate and it harboured mecA.In our study 78% of methicillin resistant M. sciuri isolates harboured mecA.The poor correlation observed in our study between phenotypic methicillin resistance and the presence of mecA in SOSA confirms challenges in using cefoxitin for screening for methicillin resistance in SOSA, as described by Yang et al. 51 and Humphries et al. 52 .
The tetracycline resistance gene tetK is the most common gene identified in tetracycline resistant staphylococcal species isolated from pigs globally 53 , and this study also identified tetK as the common gene across all staphylococcal species.The tetK gene has been reported to be plasmid-mediated 43 , and this increases the risk of spread into clinical settings.The erythromycin resistance gene ermC is the most common gene identified in erythromycin resistant staphylococcal species isolated from pigs globally, and was also identified across all Vol:.(1234567890 54 .The high clindamycin resistance rate seen in this study, in the absence of clindamycin resistance genes, might be due to the expression of erm genes 55 .Some typical human-associated S. aureus strains are also found in animals; an example is the ST1 strain 56 .In this study, 53% of S. aureus isolates from pigs belonged to ST1, this shows a possible human-animal contamination on the farms or that ST1 has become animal-adapted.This strain has been reported to cause infections in cattle and pigs in Europe 57,58 .The identification of ST398 which is the most common livestock associated strain and a novel ST8620 in pigs, humans and the environment might be an indication of a possible transmission between the selected ecological niches on the farm.
M. sciuri and S. hyicus are the two most common SOSA identified in this study.The predominance of M. sciuri ST61 and ST239 on farm A and farm B respectively, is evidence of clonal spread of different STs within the two farms.ST239 and ST61 are the most common sequence types identified amongst M. sciuri, and although these strains are not newly assigned in this study, there are no reports in literature.This demonstrates the lack of published data on the strain typing of SOSA.There is no MLST scheme available for S. hyicus, restricting their characterisation.
M. sciuri was the only species in this study with diversity of SCCmec types; S. aureus and S. epidermidis harboured one SCCmec type each.SCCmec type III was the commonest among the methicillin resistant M. sciuri isolates.This is consistent with numerous studies where SCCmec type III has been the most commonly identified SCCmec type in M. sciuri from both animals and humans 44,59,60 .It has been hypothesized that SCCmec type III originated in M. sciuri, and may have been the first SCCmec cassette to be assembled, possibly by a recombination event in a human host or a human-created environment, and later was transferred to S. aureus 61 .

Conclusion
The study identified a diversity of staphylococcal species in pigs and high rates of tetracycline resistance in pig farms in South Africa, with some evidence of possible sharing of strains and antibiotic resistance genes between pigs and humans.This highlights the need for policy makers to regulate the use antibiotics, and specifically tetracycline in pig farming to reduce the burden of AMR infections in the clinical sector in South Africa.
The study highlights the need for further research on antibiotic resistance and strain typing in SOSA in animals globally and in South Africa, which can help in designing effective antibiotic resistance surveillance and control strategies in animal husbandry.
In total, 185 isolates were subjected to AST, and all (100%) were susceptible to chloramphenicol, linezolid, rifampicin, and vancomycin.All S. chromogenes, S. hyicus, and S. haemolyticus isolates were susceptible to oxacillin.All M. sciuri and S. chromogenes isolates were non-susceptible to clindamycin, and all S. chromogenes isolates were non-susceptible to tetracycline.